Data from this and other laboratories indicate that the haloetyhl nitrosoureas react extensively with nucleic acids and that these reactions are responsible for the cytotoxic effects of these agents. This hypothesis has been strengthened by evidence that resistance of different cell lines to the nitrosoureas parallels their ability to repair DNA. The studies proposed here would attack the resistance problem by, first, identifying those specific DNA modifications which are responsible for cytotoxicity, emphasizing a search for crosslinked nucleosides since resistance has been associated with diminished DNA interstrand crosslinking. A crosslink between cytosine and guanine has recently been identified, and its mechanism of formation suggests that similar crosslinks may exist between adenine and thymine. Thus, a first objective will be to identify additional crosslinked nucleosides in DNA which has been reacted with the nitrosoureas in vitro. Then we will correlate the presence of these specific lesions with the sensitivity or resistance of various cell lines to the cytotoxic action of these agents. Lesions which are present in sensitive but absent in resistant cells would, by implication, be associated with cytotoxicity. Extracts of the resistant cell lines will be tested for their ability to remove crosslinked structures from DNA in vitro. Finally, this repair assay will be utilized to develop repair inhibitors which could restore sensitivity to the resistant cell lines.